CN109061187A - A kind of clinical medicine analysis method based on functionalized nano silica - Google Patents
A kind of clinical medicine analysis method based on functionalized nano silica Download PDFInfo
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- CN109061187A CN109061187A CN201810933887.8A CN201810933887A CN109061187A CN 109061187 A CN109061187 A CN 109061187A CN 201810933887 A CN201810933887 A CN 201810933887A CN 109061187 A CN109061187 A CN 109061187A
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- drug
- nano silica
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
Abstract
The present invention provides one kind to be based on human serum albumins functionalized nano silica (nano-SiO2) clinical medicine analysis method.This method be based on Ligand Competition principle, by drug and the drug competition of horseradish peroxidase-labeled with the nano silica (HSA-SiO of human serum albumins functionalization2) combine, the quantitative analysis to drug in clinical sample is realized after centrifugation, washing, substrate colour developing.This method have the characteristics that high accuracy, it is cheap, quickly, do not need expensive instrument, the detection for drug in clinical sample provides a new method.
Description
Technical field
The present invention relates to Pharmaceutical Analysis fields, are based on functionalized nano silica (nano- in particular to one kind
SiO2) clinical medicine analysis method.
Background technique
Currently, the assay of clinical medicine, mainly chromatography (and combined gas chromatography mass spectrometry) and immunoassay.This
A little methods all have the limitation in practical application, for example chromatography needs expensive instrument, and complicated sample pre-treatments step is taken
When, detection limit for height；And immunoassay is due to needing its corresponding antibody, leads to that batch reproducibility is poor, testing cost is very high.Cause
This, being badly in need of one, opposite pre-treatment is simple, selectivity is high, testing cost is low and time-consuming short method realization contains clinical medicine
It is fixed to measure.
Human serum albumins (HSA) is an outstanding transport protein in human serum, it and certain small-molecule drugs it
Between have a very high affinity interaction, and specificity and selectivity are also very high.Therefore, valuableness is replaced using HSA that is cheap, being easy to get
Antibody establishes the enzyme-linked ligand analysis method to clinical medicine.Compared with common immunoassays method, since HSA is instead of valuableness
Therefore antibody greatly reduces testing cost.
Nano material possesses huge specific surface area, is remarkably improved analysis efficiency.Due to the biofacies of nanometer titanium dioxide
Capacitive is good, and therefore, we use solid phase carrier of the nano silica as HSA.
Summary of the invention
The first object of the present invention is to provide a kind of human serum albumins functionalized nano silica new material (HSA-
SiO2), which is in nanoscale, is stationary phase than 96 traditional orifice plates using the enzyme-linked analysis method that it is established as stationary phase
Method have higher reaction mass transfer coefficient and stability.
The second object of the present invention is to provide a kind of HSA-SiO2Preparation method, this preparation method is in aqueous phase system
Middle reaction, a step are condensed, and step is simple, reaction condition is not harsh, easy to accomplish, are suitble to industry mass production.
The third object of the present invention is to provide a kind of new method of clinical medicine analysis, that is, uses HSA-SiO2Pass through ligand
Competition, to detect the content of drug in sample to be tested.This method have high accuracy, it is cheap, quickly, do not need expensive instrument etc.
Feature, the detection for drug in clinical sample provide a new method.
In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:
A kind of human serum albumins functionalized nano silica (HSA-SiO2) preparation method, specifically: by 1g ammonia
The Nano-meter SiO_2 of base modification2It is scattered in 10mL PBS, 50mg HSA is dissolved in 40mL PBS, is then mixed the two, at room temperature
Stir 4h.400mg 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride and 250mg N- hydroxy succinimine are molten
Liquid 2mL PBS, is then added dropwise in above-mentioned mixed liquor, stirs 12h at room temperature.Then it is centrifuged off supernatant, precipitating point
It dissipates in the skimmed milk power of 100mL 5%, at room temperature gentle agitation 4h.It is then centrifuged for, precipitating is washed three times with PBS, and freeze-drying is
?.It is put at 4 DEG C and saves backup.
The analyzing novel methods process of clinical medicine comprising:
Step a: prepare the drug (HRP-drug) of horseradish peroxidase-labeled: 0.1mM drug to be measured is dissolved in 4mL DMF
In, 0.11mM 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride is added under ice bath and stirs 15min, then
To middle addition 0.11mM N- hydroxy succinimine, it is stirred overnight under ice bath.Then reaction solution is centrifuged at 4000rpm
Supernatant is added dropwise in the horseradish peroxidase solution of 10mL 1mg/mL, under condition of ice bath with rear chamber by 5min
Temperature is lower to stir 12h.Then reaction solution is dialysed for 24 hours with PBS to get the drug for arriving horseradish peroxidase-labeled at 4 DEG C
(HRP-drug), it is put at 4 DEG C and saves backup.
Step b: drug to be measured (drug) sample and the drug (HRP-drug) of the horseradish peroxidase-labeled is mixed
It closes, concussion reaction 5-10min；Then, then the nano silica (HSA-SiO with the human serum albumins functionalization2), shake
Reaction 5-10min, centrifugation are swung, precipitating uses PBS (20mM, pH7.4) centrifuge washing three times.
Step c: finally dispersing precipitating in 1mL substrate solution, and develop the color 15min at room temperature, and the end of 0.5mL is added immediately
Only liquid detects absorbance of the supernatant at 450nm, obtains drug in sample to be tested using calibration curve method after being centrifuged
Content.
Compared with prior art, the invention has the benefit that
A. compared with chromatography (and combined gas chromatography mass spectrometry), do not need expensive instrument (only needing ultraviolet specrophotometer),
Sample pre-treatments step is simple, analyte detection process is rapid, detection limit is low.
B. compared with immunoassay, with the antibody of HAS alternatives to medication that is cheap, being easy to get, thus greatly reduce detection at
Point.
C. the introducing of nano material, substantially increases the mass tranfer coefficient of detection process, to significantly improve analysis efficiency
With the uniformity and reproducibility of analysis.
With human serum albumins functionalized nano silica (HSA-SiO2) principle to detect clinical medicine is: due to
Drug (drug) and human serum albumins (HSA) have strong ligand --- acceptor interaction, the drug in sample to be tested
(drug) be added quantitative HRP-drug competitively with HSA-SiO2In conjunction with being respectively formed drug-HSA-SiO2Compound and
drug-HRP-HSA-SiO2Compound；After centrifugation, washing, in obtained drug-HRP-HSA-SiO2The middle substrate that HRP is added
(3,3 ' 5,5 '-tetramethyl benzidines, TMB), the product of formation is blue, then uses H2SO4Reaction is terminated, the product of TMB is by indigo plant
Discoloration is yellow.Absorbance of the reaction solution at 450nm is surveyed using colorimetric method, its numerical value is brought into and is used by series standard solution
In the same resulting standard curve of detection method, the content of drug in you can get it sample to be tested.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described.
Fig. 1 is HSA-SiO2Transmission electron microscope picture；
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products that can be obtained by commercially available purchase.
Embodiment 1
Present embodiment provides a kind of nano silica (HSA-SiO of human serum albumins functionalization2) preparation side
Method:
By the amido modified Nano-meter SiO_2 of 1g2(NH2-SiO2) be scattered in 10mL PBS, 50mg HSA is dissolved in 40mL PBS
In, then the two is mixed, stirs 4h at room temperature.400mg 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride
With 250mg N- hydroxy succinimine solution 2mL PBS, then it is added dropwise in above-mentioned mixed liquor, stirs 12h at room temperature.With
After be centrifuged off supernatant, precipitating is scattered in the skimmed milk power of 100mL 5%, at room temperature gentle agitation 4h.It is then centrifuged for, sinks
Shallow lake is washed three times with PBS, is lyophilized to obtain the final product.It is put at 4 DEG C and saves backup.
Using transmission electron microscope to HSA-SiO2Material is characterized, as shown in Figure 1, it is known that its partial size in 100nm or so,
There is many protrusions on surface, be since HSA has been bonded in nano-SiO2Surface.Using thermogravimetric analyzer to HSA-SiO2
Material is characterized, by NH2-SiO2With HSA-SiO2Thermogravimetric analysis, the weight-loss curve of the two is that have apparent difference
, HSA-SiO2Reduced gravity situations obviously overweight NH2-SiO2, this is because the difference of the content of organics on the two surface.As a result,
It may indicate that HAS has successfully been coupled at nano-SiO2Surface.
Embodiment 2
Present embodiment provides a kind of preparation method of the naproxen (HRP-Naproxen) of horseradish peroxidase-labeled:
It is labeled drug with naproxen to measure naproxen (Naproxen) content.0.1mM naproxen is taken to be dissolved in 4mL
In DMF, 0.11mM 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride is added under ice bath and stirs 15min,
Then to middle addition 0.11mM N- hydroxy succinimine, it is stirred overnight under ice bath.Then by reaction solution at 4000rpm from
Supernatant is added dropwise in the horseradish peroxidase solution of 10mL 1mg/mL, then by heart 5min under condition of ice bath
12h is stirred at room temperature.Then reaction solution is dialysed for 24 hours with PBS to get the Nabumetone for arriving horseradish peroxidase-labeled at 4 DEG C
Raw (HRP-Naproxen), is put at 4 DEG C and saves backup.
It is analyzed respectively with molecular weight of the MALDI/TOF/MS to HRP and HRP-Naproxen, molecular ion peak
Respectively 43291.9809 and 44198.9537.Molecular ion peak of the HRP-Naproxen compared with HRP increases 906.9728, explanation
Naproxen small molecule is successfully coupled on HRP, and coupling ratio is 906.9728 ÷ 230.26=3.94, i.e. has been coupled about 4 on HRP
A naproxen molecule.
Embodiment 3
Present embodiment provides a kind of system of the Indomethacin (HRP-Indomethacin) of horseradish peroxidase-labeled
Preparation Method:
As different from Example 2: labeled drug is Indomethacin (Indomethacin).
Embodiment 4
Present embodiment provides a kind of preparation of the Diclofenac (HRP-Diclofenac) of horseradish peroxidase-labeled
Method:
As different from Example 2: labeled drug is Diclofenac (Diclofenac).
Embodiment 5
Present embodiment provides a kind of preparation method of the sulindac (HRP-Sulindac) of horseradish peroxidase-labeled:
As different from Example 2: labeled drug is sulindac (Sulindac).
Embodiment 6
Present embodiment provides a kind of preparation of the Gemfibrozil Capsules (HRP-Gemfibrozil) of horseradish peroxidase-labeled
Method:
As different from Example 2: labeled drug is Gemfibrozil Capsules (Gemfibrozil).
Embodiment 7
Present embodiment provides a kind of preparation of the Febuxostat (HRP-Febuxostat) of horseradish peroxidase-labeled
Method:
As different from Example 2: labeled drug is Febuxostat (Febuxostat).
Embodiment 8
Present embodiment provides a kind of detection method of naproxen (Naproxen) in urine:
Step a: sample measurement
0.5mL HRP-Naproxen liquid, concussion reaction 8min will be added into 1mL urine；Then, it is added, concussion reaction
5-10min, centrifugation, precipitating with PBS (20mM, pH 7.4) centrifuge washing three times after, disperse precipitating in 1mL substrate solution,
Develop the color 15min at room temperature, and the terminate liquid of 0.5mL is added immediately, after being centrifuged, detects absorbance of the supernatant at 450nm, adopts
The content that naproxen in sample to be tested is obtained with calibration curve method is 53.22ng/mL.
Step b: standard curve is established
The operation of similar sample measurement, does abscissa with the concentration of naproxen, and with Percentage bound B/B0, (B0 is naproxen concentration
Absorbance value when for 0ng/mL, B are the corresponding absorbance value of different naproxen concentration) be ordinate, binary once linear time
Return to obtain y=-0.013x+0.988 (R2=0.998), linear detection range 5-70ng/mL, in the range linear relationship
Well.
Step c: the verifying of method is investigated
The verifying of precision is verified by the coefficient of variation in a few days tested with tested in the daytime, result such as 1 institute of table
Show: in a few days test is the naproxen by three kinds of various concentrations (15ng/mL, 30ng/mL, 60ng/mL) of replication in 12h
Titer 9 times completions, CV is respectively 2.3%, 3.9% and 2.0%.In the daytime test be by measured respectively in seven days this three
Kind is completed, and CV is respectively 3.4%, 3.5% and 2.5%.The CV of the two is respectively less than 5%, illustrates that this method has preferable essence
Density.
The investigation result of 1 precision of table
Accuracy is verified by TIANZHU XINGNAO Capsul.First by sample replication six times, testing result is
187.23 ± 3.41ng/mL the naproxen standard solution of the concentration 80%, 100% and 120% is added into sample, i.e.,
Then 150ng/mL, 190ng/mL and 225ng/mL are measured according to the method for foundation.Its testing result is as shown in table 2: its
The rate of recovery is between 94.49%-97.95%, therefore this method has preferable accuracy, can be used for naproxen in urine
Detection.
The investigation result of 2 accuracy of table
Embodiment 9
Present embodiment provides a kind of detection method of Indomethacin (Indomethacin) in urine:
As different from Example 8: substituting HRP-Naproxen with HRP-Indomethacin.
Embodiment 10
Present embodiment provides a kind of detection method of Diclofenac (Diclofenac) in urine:
As different from Example 8: substituting HRP-Naproxen with HRP-Diclofenac.
Embodiment 11
Present embodiment provides a kind of detection method of sulindac (Sulindac) in urine:
As different from Example 8: substituting HRP-Naproxen with HRP-Sulindac.
Embodiment 12
Present embodiment provides a kind of detection method of Gemfibrozil Capsules (Gemfibrozil) in urine:
As different from Example 8: substituting HRP-Naproxen with HRP-Gemfibrozil.
Embodiment 13
Present embodiment provides a kind of detection method of Febuxostat (Febuxostat) in urine:
As different from Example 8: substituting HRP-Naproxen with HRP-Febuxostat.
Claims (7)
1. a kind of clinical medicine analysis method based on functionalized nano silica, which is characterized in that former based on Ligand Competition
Reason, through the drug competition of drug and horseradish peroxidase (HRP) label in conjunction with the nano silica of functionalization,
The quantitative analysis to drug in clinical sample is realized after centrifugation, washing, substrate colour developing.
2. a kind of clinical medicine analysis method based on functionalized nano silica according to claim 1, feature
It is, the nano silica of the functionalization is the nano silica (HSA- of human serum albumins (HSA) functionalization
SiO2)。
3. nano silica (the HSA-SiO of human serum albumins (HSA) functionalization according to claim 22) preparation side
Method, which is characterized in that obtain HAS and preparatory amido modified nano silica chemical coupling.
4. a kind of clinical medicine analysis method based on functionalized nano silica according to claim 1, feature
Be, the clinical medicine be plasma protein binding rate greater than 90% and have free carboxylic acid groups drug.
5. a kind of clinical medicine analysis method based on functionalized nano silica according to claim 4, feature
It is, the clinical medicine is naproxen, Indomethacin, Diclofenac, sulindac, Gemfibrozil Capsules or Febuxostat.
6. the drug of horseradish peroxidase (HRP) label according to claim 1, which is characterized in that by drug molecule
In free carboxy acid's base and HRP molecule in free amine group, condensation dehydration by way of, coupling gained.
7. a kind of clinical medicine analysis method based on functionalized nano silica according to claim 1, feature
It is comprising:
Step a: the nano silica (HSA-SiO of human serum albumins functionalization is prepared2)
Step b: the drug (HRP-drug) of horseradish peroxidase-labeled is prepared；
Step c: drug to be measured (drug) sample is mixed with the drug (HRP-drug) of the horseradish peroxidase-labeled, shake
Swing reaction 5-10min；Then, then the nano silica (HSA-SiO with the human serum albumins functionalization2), concussion is anti-
5-10min is answered, is centrifuged, precipitating uses PBS (20mM, pH 7.4) centrifuge washing three times.
Step d: finally dispersing precipitating in 1mL substrate solution, and develop the color 15min at room temperature, and the termination of 0.5mL is added immediately
Liquid detects absorbance of the supernatant at 450nm after being centrifuged, and obtains containing for drug in sample to be tested using calibration curve method
Amount.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110006863A (en) * | 2019-04-08 | 2019-07-12 | 中南大学湘雅三医院 | Clinical medicine analysis method based on human albumin's functionalized nano silica |
| CN110988359A (en) * | 2020-02-11 | 2020-04-10 | 中国科学院成都生物研究所 | Preparation of Au @ HIF-1 α aptamer @ Au nanoenzyme and application of Au @ HIF-1 aptamer @ Au nanoenzyme in detection of latent coronary heart disease |
-
2018
- 2018-08-16 CN CN201810933887.8A patent/CN109061187A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| QIAN-LONG WANG 等: "Development of a nano-SiO2 based enzyme-linked ligand binding assay for the determination of ibuprofen in human urine", 《TALANTA》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110006863A (en) * | 2019-04-08 | 2019-07-12 | 中南大学湘雅三医院 | Clinical medicine analysis method based on human albumin's functionalized nano silica |
| CN110988359A (en) * | 2020-02-11 | 2020-04-10 | 中国科学院成都生物研究所 | Preparation of Au @ HIF-1 α aptamer @ Au nanoenzyme and application of Au @ HIF-1 aptamer @ Au nanoenzyme in detection of latent coronary heart disease |
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Application publication date: 20181221 |